Gene Deliveryto Mammalian Cells

M E T H O D S I N M O L E C U L A R B I O L O G Y™

John M. Walker, SERIES EDITOR

268. Public Health Microbiology: Methods andProtocols, edited by John F. T. Spencer andAlicia L. Ragout de Spencer, 2004

267. Recombinant Gene Expression: Reviews andProtocols, Second Edition, edited by PaulinaBalbas and Argelia Lorence, 2004

266. Genomics, Proteomics, and Clinical Bacteriology:Methods and Reviews, edited by Neil Woodfordand Alan Johnson, 2004

265. RNA Interference, Editing, and Modification:Methods and Protocols, edited by Jonatha M.Gott, 2004

264. Protein Arrays: Methods and Protocols, ed-ited by Eric Fung, 2004

263. Flow Cytometry, Second Edition, edited byTeresa S. Hawley and Robert G. Hawley, 2004

262. Genetic Recombination Protocols, edited byAlan S. Waldman, 2004

261. Protein–Protein Interactions: Methods andApplications, edited by Haian Fu, 2004

260. Mobile Genetic Elements: Protocols and Ge-nomic Applications, edited by Wolfgang J.Miller and Pierre Capy, 2004

259. Receptor Signaling Transduction Protocols,Second Edition, edited by Gary B. Willars andR. A. John Challiss, 2004

258. Gene Expression Profiling: Methods and Pro-tocols, edited by Richard A. Shimkets, 2004

257. mRNA Processing and Metabolism: Methodsand Protocols, edited by Daniel R. Schoenberg,2004

256. Bacterial Artifical Chromosomes, Volume 2:Functional Studies, edited by Shaying Zhaoand Marvin Stodolsky, 2004

255. Bacterial Artifical Chromosomes, Volume 1:Library Construction, Physical Mapping, andSequencing, edited by Shaying Zhao andMarvin Stodolsky, 2004

254. Germ Cell Protocols, Volume 2: MolecularEmbryo Analysis, Live Imaging, Transgenesis,and Cloning, edited by Heide Schatten, 2004

253. Germ Cell Protocols, Volume 1: Sperm andOocyte Analysis, edited by Heide Schatten,2004

252. Ribozymes and siRNA Protocols, SecondEdition, edited by Mouldy Sioud, 2004

251. HPLC of Peptides and Proteins: Methodsand Protocols, edited by Marie-Isabel Aguilar,2004

250. MAP Kinase Signaling Protocols, edited byRony Seger, 2004

249. Cytokine Protocols, edited by Marc De Ley,2004

248. Antibody Engineering: Methods and Protocols,edited by Benny K. C. Lo, 2004

247. Drosophila Cytogenetics Protocols, edited byDaryl S. Henderson, 2004

246. Gene Delivery to Mammalian Cells: Volume2: Viral Gene Transfer Techniques, edited byWilliam C. Heiser, 2004

245. Gene Delivery to Mammalian Cells: Volume1: Nonviral Gene Transfer Techniques, editedby William C. Heiser, 2004

244. Protein Purification Protocols, Second Edi-tion, edited by Paul Cutler, 2004

243. Chiral Separations: Methods and Protocols,edited by Gerald Gübitz and Martin G.Schmid, 2004

242. Atomic Force Microscopy: Biomedical Meth-ods and Applications, edited by Pier CarloBraga and Davide Ricci, 2004

241. Cell Cycle Checkpoint Control Protocols,edited by Howard B. Lieberman, 2004

240. Mammalian Artificial Chromosomes:Methods and Protocols, edited by VittorioSgaramella and Sandro Eridani, 2003

239. Cell Migration in Inflammation and Immu-nity: Methods and Protocols, edited by DanieleD’Ambrosio and Francesco Sinigaglia, 2003

238. Biopolymer Methods in Tissue Engineering,edited by Anthony P. Hollander and Paul V.Hatton, 2003

237. G Protein Signaling: Methods and Protocols,edited by Alan V. Smrcka, 2003

236. Plant Functional Genomics: Methods andProtocols, edited by Erich Grotewold, 2003

235. E. coli Plasmid Vectors: Methods and Appli-cations, edited by Nicola Casali and AndrewPreston, 2003

234. p53 Protocols, edited by Sumitra Deb andSwati Palit Deb, 2003

233. Protein Kinase C Protocols, edited byAlexandra C. Newton, 2003

232. Protein Misfolding and Disease: Principlesand Protocols, edited by Peter Bross and NielsGregersen, 2003

231. Directed Evolution Library Creation: Meth-ods and Protocols, edited by Frances H.Arnold and George Georgiou, 2003

230. Directed Enzyme Evolution: Screening andSelection Methods, edited by Frances H.Arnold and George Georgiou, 2003

M E T H O D S I N M O L E C U L A R B I O L O G Y™

Gene Deliveryto Mammalian Cells

Volume 1: Nonviral Gene Transfer Techniques

Edited by

William C. HeiserBio-Rad Laboratories

Hercules, CA

Totowa, New JerseyHumana Press

© 2004 Humana Press Inc.999 Riverview Drive, Suite 208Totowa, New Jersey 07512

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Cover illustration: Foreground graphic from Fig. 1A,B in Chapter 14 (Volume 1) "Delivery of DNA to Skinby Particle Bombardment," by Shixia Wang, Swati Joshi, and Shan Lu. Background graphic from Fig. 3 inChapter 33 (Volume 2) "Retrovirus-Mediated Gene Transfer to Tumors: Utilizing the Replicative Power ofViruses to Achieve Highly Efficient Tumor Transduction In Vivo," by Christopher R. Logg and NoriyukiKasahara.

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Library of Congress Cataloging in Publication DataGene delivery to mammalian cells / edited by William C. Heiser. p. ; cm. -- (Methods in molecular biology ; 245-246) Includes bibliographical references and indexes. Contents: v. 1. Nonviral gene transfer techniques -- v. 2. Viral gene transfer techniques. ISBN 1-58829-086-7 (v. 1 : alk. paper) -- ISBN 1-58829-095-6 (v. 2 : alk. paper) ISSN 1064-3745 1. Transfection. 2. Animal cell biotechnology. I. Heiser, William C. II. Series: Methods in molecularbiology (Totowa, N.J.) ; 245-246. [DNLM: 1. Cloning, Molecular. 2. Gene Transfer Techniques. 3. Cells, Cultured--cytology. 4. GeneTargeting--methods. 5. Mammals--genetics. QH 442.2 G327 2004] QH448.4.G42 2004 571.9'64819--dc21

2003006980

v

Preface

The efficiency of delivering DNA into mammalian cells has increased tre-mendously since DEAE dextran was first shown to be capable of enhancingtransfer of RNA into mammalian cells in culture. Not only have other chemicalmethods been developed and refined, but also very efficient physical and viraldelivery methods have been established. The technique of introducing DNAinto cells has developed from transfecting tissue culture cells to deliveringDNA to specific cell types and organs in vivo. Moreover, two important areasof biology—assessment of gene function and gene therapy—require success-ful DNA delivery to cells, driving the practical need to increase the efficiencyand efficacy of gene transfer both in vitro and in vivo.

These two volumes of the Methods in Molecular BiologyTM series, Gene Deliv-ery to Mammalian Cells, are designed as a compendium of those techniques thathave proven most useful in the expanding field of gene transfer in mammaliancells. It is intended that these volumes will provide a thorough background onchemical, physical, and viral methods of gene delivery, a synopsis of the myriadtechniques currently available to introduce genes into mammalian cells, as wellas a practical guide on how to accomplish this. It is my expectation that it willbe useful to the novice in the field as well as to the scientist with expertise ingene delivery.

Volume 1: Nonviral Gene Transfer Techniques discusses delivery of DNA intocells by nonviral means, specifically chemical and physical methods. Volume 2:Viral Gene Transfer Techniques details procedures for delivering genes into cellsusing viral vectors. Each volume is divided into sections; each section beginswith a chapter that provides an overview of the basis behind the deliverysystem(s) described in that section. The succeeding chapters provide detailedprotocols for using these techniques to deliver genes to cells in vitro and invivo. Many of these techniques have only been in practice for a few years andare still being refined and updated. Some are being used not only in basic sci-ence, but also in gene therapy applications.

I wish to express my thanks to all of the authors who made Gene Delivery toMammalian Cells: Volume 1: Nonviral Gene Transfer Techniques and Volume2: Viral Gene Transfer Techniques possible. I would especially like to thankthose who contributed the overview chapter to each section. They providedinvaluable discussions, suggestions, and assistance on organizing those sec-

vi Preface

tions. I would particularly like to mention Joanne Douglas, Tom Daly, and BillGoins for their suggestions on topics and authors, Dexi Liu and Shan Lu fortheir helpful discussions, and Mark Jaroszeski for his suggestions on organiz-ing the entire editing process.

William C. Heiser

vii

Contents

Preface ..............................................................................................................vContributors .................................................................................................. xiii

PART I. DELIVERY USING CHEMICAL METHODS

1. Chemical Methods for DNA Delivery: An OverviewDexi Liu, Evelyn F. Chiao, and Hui Tian ............................................... 3

2. Gene Transfer into Mammalian Cells Using Calcium Phosphateand DEAE-Dextran

Gregory S. Pari and Yiyang Xu ........................................................... 253. DNA Delivery to Cells in Culture Using Peptides

Lei Zhang, Nicholas Ambulos, and A. James Mixson ......................... 334. DNA Delivery to Cells in Culture Using PNA Clamps

Todd D. Giorgio and Shelby K. Wyatt ................................................ 535. Dendrimer-Mediated Cell Transfection In Vitro

James R. Baker, Jr., Anna U. Bielinska,and Jolanta F. Kukowska-Latallo .................................................... 67

6. DNA Delivery to Cells in Culture Using Cationic LiposomesShelby K. Wyatt and Todd D. Giorgio ................................................ 83

7. Formulation of Synthetic Gene Delivery Vectorsfor Transduction of the Airway Epithelium

John Marshall, Nelson S. Yew, and Seng H. Cheng ............................ 958. Cationic Liposome-Mediated DNA Delivery

to the Lung EndotheliumYoung K. Song, Guisheng Zhang, and Dexi Liu ................................ 115

9. Delivery of DNA to Tumor Cells Using Cationic LiposomesDuen-Hwa Yan, Bill Spohn, and Mien-Chie Hung ........................... 125

10. Delivery of Transposon DNA to Lungs of MiceUsing Polyethyleneimine-DNA Complexes

Lalitha R. Belur and R. Scott McIvor ................................................ 137

PART II. DELIVERY USING PHYSICAL METHODS

11. Gene Delivery Using Physical Methods: An OverviewTe-hui W. Chou, Subhabrata Biswas, and Shan Lu .......................... 147

12. Gene Delivery to Mammalian Cells by MicroinjectionRobert King ....................................................................................... 167

viii Contents

13. Delivery of DNA to Cells in Culture Using ParticleBombardment

William C. Heiser .............................................................................. 17514. Delivery of DNA to Skin by Particle Bombardment

Shixia Wang, Swati Joshi, and Shan Lu............................................. 18515. Biolistic Transfection of Cultured Organotypic Brain Slices

A. Kimberley McAllister .................................................................... 19716. Efficient Electroporation of Mammalian Cells in Culture

Peter A. Barry.................................................................................... 20717. Delivery of DNA to Skin by Electroporation

Nathalie Dujardin and Véronique Préat ........................................... 21518. In Vivo DNA Electrotransfer in Skeletal Muscle

Guenhaël Sanz, Saulius Satkauskas, and Lluis M. Mir ...................... 22719. Electrically Mediated Plasmid DNA Delivery to Solid

Tumors In VivoMark J. Jaroszeski, Loree C. Heller, Richard Gilbert,

and Richard Heller ....................................................................... 23720. Hydrodynamic Delivery of DNA

Joseph E. Knapp and Dexi Liu ........................................................... 24521. Naked DNA Gene Transfer in Mammalian Cells

Guofeng Zhang, Vladimir G. Budker, James J. Ludtke,and Jon A. Wolff ........................................................................... 251

22. Microparticle Delivery of Plasmid DNA to MammalianCells

Mary Lynne Hedley and Shikha P. Barman ...................................... 26523. DNA Delivery to Cells in Culture Using Ultrasound

Thomas P. McCreery, Robert H. Sweitzer,and Evan C. Unger ........................................................................ 287

24. DNA Delivery to Cells In Vivo by UltrasoundThomas P. McCreery, Robert H. Sweitzer, Evan C. Unger,

and Sean Sullivan .......................................................................... 293Index ............................................................................................................ 299

ˆ

ix

CONTENTS OF THE COMPANION VOLUME

Volume 2: Viral Gene Transfer Techniques

PART I. DELIVERY USING ADENOVIRUSES

1. Adenovirus-Mediated Gene Delivery: An OverviewJoanne T. Douglas

2. DNA Delivery to Cells in Culture: Generation of AdenoviralLibraries for High-Throughput Functional Screening

Miroslava Ogorelkova, Seyyed Mehdy Elahi, David Gagnon,and Bernard Massie

3. Adenovirus-Mediated Gene Delivery to Skeletal MuscleJoanne T. Douglas

4. Delivery of Adenoviral DNA to Mouse LiverSheila Connelly and Christine Mech

5. Delivery of DNA to Lung Airway EpitheliumDaniel J. Weiss

6. Delivery of DNA to Pulmonary Endothelium Using AdenoviralVectors

Paul N. Reynolds7. Gene Transfer to Brain and Spinal Cord Using Recombinant

Adenoviral VectorsJoseph M. Alisky and Beverly L. Davidson

8. Adenovirus-Mediated Gene Transfer to Tumor CellsManel Cascalló and Ramon Alemany

9. Adenovirus-Mediated Gene Delivery to Dendritic CellsLaura Timares, Joanne T. Douglas, Bryan W. Tillman,

Victor Krasnykh, and David T. Curiel

PART II. DELIVERY USING ADENO-ASSOCIATED VIRUSES

10. Overview of Adeno-Associated Viral VectorsThomas M. Daly

11. AAV Vector Delivery to Cells in CultureAndrew Smith, Roy Collaco, and James P. Trempe

12. AAV-Mediated Gene Transfer to Skeletal MuscleRoland W. Herzog

13. AAV-Mediated Gene Transfer to the LiverThomas M. Daly

x Contents of Companion Volume

14. AAV-Mediated Gene Transfer to Mouse LungsChristine L. Halbert and A. Dusty Miller

15. Gene Delivery to the Mammalian Heart Using AAV VectorsDanny Chu, Patricia A. Thistlethwaite, Christopher C. Sullivan,

Mirta S. Grifman, and Matthew D. Weitzman16. Gene Delivery to the Mouse Brain with Adeno-Associated Virus

Marco A. Passini, Deborah J. Watson, and John H. Wolfe17. Delivery of DNA to Tumor Cells In Vivo Using

Adeno-Associated VirusSelvarangan Ponnazhagan and Frank Hoover

18. Gene Delivery to Human and Murine Primitive HematopoieticStem and Progenitor Cells by AAV2 Vectors

Arun Srivastava

PART III. DELIVERY USING HERPES SIMPLEX VIRUSES

19. Delivery Using Herpes Simplex Virus: An OverviewWilliam F. Goins, Darren Wolfe, David M. Krisky, Qing Bai,

Ed A. Burton, David J. Fink, and Joseph C. Glorioso20. Gene Transfer to Skeletal Muscle Using Herpes Simplex

Virus-Based VectorsBaohong Cao and Johnny Huard

21. Delivery of Herpes Simplex Virus-Based Vectorsto the Nervous System

James R. Goss, Atsushi Natsume, Darren Wolfe, Marina Mata,Joseph C. Glorioso, and David J. Fink

22. Gene Transfer to Glial Tumors Using Herpes Simplex VirusAjay Niranjan, Darren Wolfe, Wendy Fellows, William F. Goins,

Joseph C. Glorioso, Douglas Kondziolka,and L. Dade Lunsford

23. Delivery of Herpes Simplex Virus-Based Vectors to Stem CellsDarren Wolfe, James B. Wechuck, David M. Krisky, Julie P. Goff,

William F. Goins, Ali Ozuer, Michael E. Epperly,Joel S. Greenberger, David J. Fink, and Joseph C. Glorioso

PART IV. DELIVERY USING BACULOVIRUSES

24. Baculovirus-Mediated Gene Delivery into Mammalian CellsRaymond V. Merrihew, Thomas A. Kost,

and J. Patrick Condreay

Contents of Companion Volume xi

PART V. DELIVERY USING LENTIVIRUSES

25. Gene Delivery by Lentivirus Vectors: An OverviewTal Kafri

26. Lentiviral Vectors for the Delivery of DNA into Mammalian CellsRoland Wolkowicz, Garry P. Nolan, and Michael A. Curran

27. Stable Gene Delivery to CNS Cells Using Lentiviral VectorsDeborah J. Watson, Brian A. Karolewski, and John H. Wolfe

28. Gene Delivery to Hematopoietic Stem Cells UsingLentiviral Vectors

Hiroyuki Miyoshi29. Delivery of Genes to the Eye Using Lentiviral Vectors

Masayo Takahashi30. Lentiviral Transduction of Human Dendritic Cells

Roland Schroers and Si-Yi Chen

PART VI. DELIVERY USING RETROVIRUSES

31. Gene Transfer by Retroviral Vectors: An OverviewNikunj Somia

32. Gene Delivery to Cells in Culture Using RetrovirusesNikunj Somia

33. Retrovirus-Mediated Gene Transfer to Tumors: Utilizing theReplicative Power of Viruses to Achieve Highly EfficientTumor Transduction In Vivo

Christopher R. Logg and Noriyuki Kasahara34. Delivery of Genes to Hematopoietic Stem Cells

Masafumi Onodera

PART VII. DELIVERY USING ALPHAVIRUSES

35. Delivery and Expression of Heterologous Genes in Mammalian CellsUsing Self-Replicating Alphavirus Vectors

Gunilla B. Karlsson and Peter Liljeström

Contributors

NICHOLAS AMBULOS • Department of Microbiology, University of Maryland,Baltimore, MD

JAMES R. BAKER, JR. • Department of Internal Medicine and Center forBiologic Nanotechnology, The University of Michigan Health System,Ann Arbor, MI

SHIKHA P. BARMAN • ZYCOS Inc., Lexington, MAPETER A. BARRY • Center for Comparative Medicine, University of

California, Davis, CALALITHA R. BELUR • Institute of Human Genetics, University of Minnesota,

Minneapolis, MNANNA U. BIELINSKA • Department of Internal Medicine and Center for

Biologic Nanotechnology, The University of Michigan Health System,Ann Arbor, MI

SUBHABRATA BISWAS • Department of Medicine, University of MassachusettsMedical School, Worcester, MA

VLADIMIR G. BUDKER • Departments of Pediatrics and Medical Genetics,University of Wisconsin, Madison, WI

SENG H. CHENG • Genzyme Corporation, Framingham, MAEVELYN F. CHIAO • Department of Pharmaceutical Sciences, School of

Pharmacy, University of Pittsburgh, Pittsburgh, PATE-HUI W. CHOU • Department of Medicine, University of Massachusetts

Medical School, Worcester, MANATHALIE DUJARDIN • Unité de Pharmacie galénique, Université Catholique

de Louvain, Brussels, BelgiumRICHARD GILBERT • Department of Chemical Engineering, College of

Engineering, University of South Florida, Tampa, FLTODD D. GIORGIO • Departments of Biomedical Engineering and Chemical

Engineering, Vanderbilt University, Nashville, TNMARY LYNNE HEDLEY • ZYCOS Inc., Lexington, MAWILLIAM C. HEISER • Life Science Group, Bio-Rad Laboratories, Hercules,

CALOREE C. HELLER • Department of Surgery, College of Medicine, University

of South Florida, Tampa, FLRICHARD HELLER • Department of Surgery, College of Medicine, University

of South Florida, Tampa, FL

xiii

MIEN-CHIE HUNG • Department of Molecular and Cellular Oncology,University of Texas, M. D. Anderson Cancer Center, Houston, TX

MARK J. JAROSZESKI • Department of Chemical Engineering, College ofEngineering, University of South Florida, Tampa, FL

SWATI JOSHI • Department of Medicine, University of Massachusetts MedicalSchool, Worcester, MA

ROBERT KING • Life Science Group, Bio-Rad Laboratories, Hercules, CAJOSEPH E. KNAPP • Department of Pharmaceutical Sciences, School of

Pharmacy, University of Pittsburgh, Pittsburgh, PAJOLANTA F. KUKOWSKA-LATALLO • Department of Internal Medicine and

Center for Biologic Nanotechnology, The University of Michigan HealthSystem, Ann Arbor, MI

DEXI LIU • Department of Pharmaceutical Sciences, School of Pharmacy,University of Pittsburgh, Pittsburgh, PA

SHAN LU • Department of Medicine, University of Massachusetts MedicalSchool, Worcester, MA

JAMES J. LUDTKE • Waisman Center, University of Wisconsin, Madison, WIJOHN MARSHALL • Genzyme Corporation, Framingham, MAA. KIMBERLEY MCALLISTER • Center for Neuroscience and Department of

Neurology, University of California, Davis, CATHOMAS P. MCCREERY • ImaRx Therapeutics Inc., Tucson, AZR. SCOTT MCIVOR • Institute of Human Genetics, University of Minnesota,

Minneapolis, MNLLUIS M. MIR • C.N.R.S., Institut Gustave-Roussy, Villejuif, FranceA. JAMES MIXSON • Department of Pathology, University of Maryland,

Baltimore, MDGREGORY S. PARI • Department of Microbiology, University of Nevada,

Reno, NVVÉRONIQUE PRÉAT • Unité de Pharmacie galénique, Université Catholique de

Louvain, Brussels, BelgiumGUENHAËL SANZ • Institut National de la Recherche Agronomique, Jouy-en-

Josas, FranceSAULIUS SATKAUSKAS • Department of Biology, Vytautas Magnus University,

Kaunas, LithuaniaYOUNG K. SONG • Department of Pharmacology, School of Medicine,

University of Pittsburgh, Pittsburgh, PABILL SPOHN • Department of Molecular and Cellular Oncology, The

University of Texas, M.D. Anderson Center, Houston, TXSEAN SULLIVAN • Department of Pharmceutics, University of Florida,

Gainsville, FL

xiv Contributors

ˆ

ROBERT H. SWEITZER • ImaRx Therapeutics Inc., Tucson, AZHUI TIAN • Department of Pharmaceutical Sciences, School of Pharmacy,

University of Pittsburgh, Pittsburgh, PAEVAN C. UNGER • Department of Radiology, University of Arizona Health

Sciences Center, Tucson, AZ, and ImaRx Therapeutics Inc., Tucson, AZSHIXIA WANG • Department of Medicine, University of Massachusetts

Medical School, Worcester, MAJON A. WOLFF • Departments of Pediatrics and Medical Genetics and

Waisman Center, University of Wisconsin, Madison, WISHELBY K. WYATT • Department of Biomedical Engineering, Vanderbilt

University, Nashville, TNYIYANG XU • Department of Microbiology, University of Nevada, Reno, NVDUEN-HWA YAN • Department of Molecular and Cellular Oncology, The

University of Texas, M.D. Anderson Center, Houston, TXNELSON S. YEW • Genzyme Corporation, Framingham, MAGUISHENG ZHANG • Department of Pharmaceutical Sciences, School of

Pharmacy, University of Pittsburgh, Pittsburgh, PAGUOFENG ZHANG • Waisman Center, University of Wisconsin, Madison, WILEI ZHANG • Department of Pathology, University of Maryland, Baltimore,

MD

Contributors xv

From: Methods in Molecular Biology, vol. 245:Gene Delivery to Mammalian Cells: Vol. 1: Nonviral Gene Transfer Techniques

Edited by: W. C. Heiser © Humana Press Inc., Totowa, NJ

25

2

Gene Transfer into Mammalian Cells Using Calcium Phosphate and DEAE-Dextran

Gregory S. Pari and Yiyang Xu

1. IntroductionSimplicity and cost are just two of the factors that have sustained the popu-

larity of calcium phosphate and, to a lesser extent, DEAE-dextran transfectionmethods. However, notwithstanding these factors, the calcium phosphatemethod, especially use of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid(the BES variation), has proven to be the only method sufficient for the co-transfection of multiple plasmids into a wide variety of cell types. Although notas widely used today, DEAE-dextran-mediated transfection is a highly repro-ducible method for transient expression of a foreign gene.

The DEAE-dextran-mediated transfection method was widely used in theearly to mid-1980s because of the simplicity, efficiency, and reproducibility ofthe procedure (1–5). One major drawback of this method is the poor efficiencyin forming stable cell lines. In addition, cellular toxicity is high because it isnecessary to expose the cells to dimethyl sulfoxide (DMSO). Consequently,DEAE-dextran-mediated transfection has fallen out of favor with many inves-tigators, giving way mostly to lipid-mediated transfection. However, becauselipid-mediated transfection can be costly and inefficient in some cell types,many laboratories may want to consider the DEAE-dextran method. Some in-vestigators have found DEAE-dextran-mediated transfection to be highly ef-fective in certain cell lines. Several reports demonstrated that this is the methodof choice for delivering DNA to primary cultured human macrophages (6,7). Inaddition, it appears that DEAE-dextran enhances the transfection efficiency ofmammalian cells when using electroporation (8).

In contrast to DEAE-dextran, the calcium phosphate co-precipitation proce-dure has remained a popular method to efficiently deliver DNA to a wide vari-ety of cell types. The main advantage of calcium phosphate DNA transfectionis the high efficiency for the generation of constitutively expressing cell lines.Calcium phosphate is the method of choice for the simultaneous transfection ofmultiple plasmids. In our laboratory, we routinely co-transfect as many as 12different plasmid constructs at the same time into mammalian cells. PlasmidDNA to be transfected must be of the highest purity, usually only double-bandedCsCl DNA is used for transfection.

The original calcium phosphate method used a HEPES-based buffer sys-tem (9). This method is simple to use, but is limited in the range of cell linesthat can be efficiently transfected. Many variations of the HEPES-based systemexist, and some have optimized this method for a particular cell type (10). Avariation of the original calcium phosphate transfection method, one that usesBES buffer, has emerged as a very versatile and highly efficient transfectionmethod (11). The BES method uses a different buffer system in which the pHis lower than the HEPES-based procedure. A lower pH, coupled with incuba-tion in a reduced CO2 atmosphere for 15 h, allows the DNA-calcium phosphateprecipitate to form slowly on the cells. This results in a significant increase inthe efficiency of transfection and a higher percentage of stably expressing celllines than the HEPES-based procedure. Co-transfection efficiencies are alsomuch higher using the BES method versus the original HEPES-based buffertransfection method. This feature is of particular importance to establish a co-transfection replication assay (12,13). Because of these advantages, we presentonly the BES method in this chapter.

The following are representative protocols for DEAE-dextran transfection ofadherent and suspension cells, as well as a protocol for the BES method for cal-cium phosphate-mediated transfection.

2. Materials2.1. DEAE-Dextran Transfection

1. Tris-buffered saline (TBS): Prepare the following sterile solutions: SolutionA: 80 g/L NaCl, 3.8 g/L KCl, 2 g/L Na2HPO4, 30 g/L Tris base. Adjust pH to7.5. Solution B: 15 g/L CaCl2, 10 g/L MgCl2. Filter-sterilize both solutionsand store at �20°C. To make 100 mL of working solution, add 10 mL of So-lution A to 89 mL of water and then add 1 mL of Solution B, filter-sterilizeand store at 4°C.

2. Suspension Tris-buffered saline (STBS): 25 mM Tris-HCl, pH 7.4, 137 mMNaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, 0.5 mM MgCl2. Dis-solve in distilled H2O and filter-sterilize.

26 Pari and Xu

3. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 4.3 mMNa2HPO4, 1.4 mM KH2PO4, pH 7.3.

4. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%fetal bovine serum (FBS).

5. DEAE-dextran: 10 mg/mL in TBS.6. 10% DMSO.7. For suspension cultures, cells are grown in RPMI 1640 medium supple-

mented with 10–20% FBS.

2.2. Calcium Phosphate Co-Precipitation

1. DMEM supplemented with 10% FBS.2. CsCl-purified double-banded DNA.3. 2.5 M CaCl2 filter sterilized through a 0.45-lm filter.4. 2X BES-buffered saline (BBS): 50 mM N,N-bis(2-hydroxyethyl)-2-

aminoethanesulfonic acid, 1.5 mM Na2HPO4, 280 mM NaCl. Adjust pH to6.95 with 1 N NaOH (see Note 1).

5. 35°C 3% CO2 humidified incubator.

3. Methods3.1. DEAE-Dextran Methods

Two DEAE-dextran methods are commonly used. The first is the basic pro-tocol, which can be used on all anchorage-dependent cells. The second can beused on cells that normally grow in suspension or with anchorage-dependentcells that have been trypsinized and are in suspension. This procedure may in-crease transfection efficiency in some cells. For adherent cells, it is advisable totry the basic protocol first, then if transfection efficiency is low, try the suspen-sion procedure.

3.1.1. Anchorage-Dependent Cells

1. Plate 5 � 105 cells in a 10 cm tissue culture dish (see Note 2). Cells shouldbe plated at least 24 h before transfection and should be no more than40–60% confluent.

2. For each plate of cells to be transfected, ethanol precipitate 5 lg of DNAin a 1.5-mL microcentrifuge tube and resuspend the pellet in 40 lL of TBS.If the same DNA is used for multiple plates, precipitate all the DNA in onetube. Ethanol precipitation sterilizes DNA.

3. Remove media from the cells and wash cells with 10 mL of PBS. After wash-ing, add 4 mL (for a 10-cm dish) of DMEM supplemented with 10% FBS.

4. Aliquot 80 lL of DEAE-dextran into 1.5-mL microfuge tubes and warm to37°C in a water bath. Add the resuspended DNA (5 lg of DNA per 80 lLof DEAE-dextran) slowly to the tube while vortexing gently.

Calcium Phosphate and DEAE-Dextran 27

5. Add 120 lL of the DNA/DEAE-dextran mixture to the plate in a dropwisefashion using a 200 lL pipet tip. Swirl the plate after each drop is appliedto ensure that the mixture is distributed evenly (see Note 3).

6. Incubate the plates for 4 h in a 37°C incubator with a 5% CO2 atmosphere.This incubation time can be shortened for some cell types.

7. Remove the medium. At this point, the cells may look a little sick but thisis normal.

8. Add 5 mL of 10% DMSO in PBS. Incubate for 1 min at room temperature.Remove the DMSO and wash the cells with 5 mL of PBS. Replace the PBSwith 10 mL of DMEM supplemented with 10% FBS.

3.1.2. Cells in Suspension

1. Ethanol precipitate DNA and resuspend the pellet in 500 lL of STBS (seeSubheading 2). Use 10 lg of DNA per 2 � 107 cells. Cells can be eithernormally growing suspension cells, for example B-cells, or trypsinized an-chorage-dependent cells.

2. Pellet cells in a 50-mL conical centrifuge tube.3. Resuspend cells in 5 mL of STBS and re-pellet as in step 2.4. Prepare a 2X solution of DEAE-dextran (200–1000 lg/mL) in STBS and

add 500 lL of this solution to 500 lL of the DNA resuspended in 500 lL ofSTBS from step 1. Mix well. Resuspend pelleted cells with this DEAE-dex-tran/DNA solution. Use a final concentration of 100–500 lg/mL of DEAE-dextran.

5. Incubate cells in a CO2 incubator for 30–90 min. Tap cells occasionally tokeep them from clumping. Incubation times vary and should be determinedexperimentally.

6. Add DMSO to cells dropwise to a final concentration of 10%, mix wellwhile adding.

7. Incubate cell with DMSO for 2–3 min. Add 15 mL of STBS to cell.8. Pellet cells, wash with 10 mL of STBS and pellet again. Wash cells in

medium with serum and pellet. After this centrifugation, resuspend cells incomplete medium (RPMI plus 10–20% FBS). If cells are normally anchor-age-dependent, re-plate on a 10 cm dish or in a 75-cm2 flask. If cells are nor-mally grown in suspension, incubate cells in normal growth media in 25-cm2

flasks. The onset of expression from transfected plasmids varies dependingon cell type. Usually expression begins between 24–48 h post-transfection.

3.2. Calcium Phosphate Co-Precipitation Method

Like DEAE-dextran transfection, two calcium phosphate transfection meth-ods are routinely used: a HEPES-based method and a BES buffer method. Bothare good for transient expression, but the BES-buffer procedure is much moreefficient for making established constitutively expressing cell lines, in some

28 Pari and Xu

cells 50% efficiency can be achieved. In addition, this procedure works betteron a wider variety of cell types, is excellent for co-transfection, and is easier toperform than the traditional HEPES-based method. Because the BES method isso versatile and offers these advantages, this method is presented here.

1. Plate approx 5 � 105 cells on a 10-cm tissue culture dish 24 h before trans-fection. The cells should be no more than 50% confluent for making estab-lished cell lines and about 70% confluent for transient expression. Smallerplates (e.g., 6 cm) may be used and in some cases this is actually sufficientand easier.

2. Dilute the 2.5 M CaCl2 solution to 0.25 M with sterile water.3. Precipitate plasmid DNA in 17 � 100 mm polypropylene tubes as follows:

Add 20–30 lg of plasmid DNA per tube (Falcon # 2058) or 10–20 lg for a6-cm plate (see Note 4). For transfecting cells in a 100-mm dish or in two60-mm dishes, add 20–30 lg of DNA to the tube followed, in order, by 500ll of 0.25 M CaCl2, then 500 lL of 2X BBS. Use one-half of this mixture oneach plate when using 60-mm dishes. For transfecting cells in one 60-mmdish, add 10–20 lg of DNA followed, in order, by 250 lL of 0.25 M CaCl2,then 250 ll of 2X BBS. In both cases, mix well and incubate at room tem-perature for 10–20 min (see Note 5).

4. Add the calcium phosphate/DNA mixture to cells in a dropwise fashion,swirling the plate after each drop. Incubate the cells overnight in a 35°C 3%CO2 incubator (see Note 6).

5. Wash cells twice with 5 mL of PBS, then add 10 mL of DMEM with 10%FBS. Incubation of cells from this point on is done in a 5% CO2 37°C in-cubator.

6. For transient expression, harvest cells 48 h post-transfection. For selectionof stably integrated expression clones, split cells (1:10) 48 h post-transfec-tion into selection medium. For co-transfection see Note 7. Many investi-gators have used the BES method to elucidate genes required for viral DNAreplication. The BES method is well-suited for this purpose because of thehigh co-transfection efficiency (see Note 8).

4. Notes1. BBS pH is critical. Make three solutions ranging in pH from 6.93–6.98.

Usually a visual inspection of cells after an overnight transfection will in-dicate which BBS mixture and DNA concentration works best. A coarseand clumpy precipitate will form when DNA concentrations are too low, afine, almost invisible precipitate will form when DNA concentration are toohigh. An even granular precipitate forms when the concentration is justright. This usually correlates to the highest level of gene expression or for-mation of stably integrated cell lines.

Calcium Phosphate and DEAE-Dextran 29

30 Pari and Xu

Fig. 1. Cotransfection of 11 plasmids using BES calcium phosphate coprecipitation.(A) Replication assay. Human foreskin fibroblasts (HFF) were transfected with 10 plas-mids encoding human cytomegalovirus (HCMV) replication genes along with a plas-mid encoding the cloned origin of lytic replication (oriLyt). Total cellular DNA was har-vested 5 d post-transfection and cleaved with EcoRI and DpnI. DpnI, a four base-pairrecognition enzyme, will cleave input DNA (unreplicated DNA) multiple times andEcoRI will linearize the HCMV oriLyt. Replicated plasmid is DpnI-resistant and is in-dicated by the arrow. DNA is separated on an agarose gel and hybridized with the par-ent plasmid vector. Lanes: 1, All required plasmids plus HCMV oriLyt; 2, omission ofone plasmid required for oriLyt replication. (B) HCMV replication compartment for-mation requires cotransfection of essential replication proteins. Cotransfections in-cluded a replication protein fused in frame with EGFP. HFF cells were transfected with10 plasmids encoding HCMV replication proteins along with a plasmid encoding areplication protein fused in-frame with EGFP. Transfected cells were fixed and visual-ized using a confocal microscope. Panel 1: cotransfections were performed the same asin A sample number 1, cells were fixed and visualized using a confocal microscope.

2. Smaller dishes may be used; adjust the number of cells and DNA used pro-portionally. Higher densities of some cell types may be necessary toachieve good transfection efficiencies. If cell death is too high owing to thetoxicity of DEAE, then try plating cells at a higher density.

3. It may be necessary to determine the optimal concentration of DEAE-dex-tran needed for good transfection efficiencies. Vary the volume of TBS usedto resuspend DNA and the amount of DEAE-dextran. For example:

DNA in TBS, lL DEAE-dextran, mg/mL

80 16040 8020 40

4. Only high-quality plasmid DNA will work. Use only double-banded CsClpurified DNA. Carrier DNA is not necessary and actually will decrease ef-ficiency. Also, linear DNA does not transfect well.

5. At this point no precipitate should be visible. Use three different concen-trations of DNA to help identify the DNA concentration necessary for op-timal transfection.

6. CO2 level is critical. Measure the level with a Fyrite gas analyzer. Temper-ature is somewhat less critical. A 37°C incubator can be used.

7. When performing cotransfections, vary the amount of effector plasmid inrelation to the other plasmids in the mix keeping the total amount of DNAthe same. The ratio of plasmids used in the mixture can be the differencebetween success and failure. We routinely find that a higher concentrationof effector plasmid in the mix yields better results. We commonly use thiscotransfection method to assay the level of promoter activity effected bycertain vital transactivators.

8. The co-transfection-replication assay involves the co-transfection of sev-eral different plasmids (in the case of HCMV 11 different plasmids) eachencoding a gene required for DNA replication. Depending on the numberof plamids in the transfection mixture, vary the amount of transactivatorsand effector plasmid. As many as 11 plasmids can be tranfected at one time.Each plasmid can contain one or many genes required for replication of acloned origin of DNA replication (Fig. 1).

Calcium Phosphate and DEAE-Dextran 31

Panel 2: cotransfections were the same as in (A), sample number 2, in which one plas-mid encoding an essential protein was omitted from the transfection mixture. Inclusionof all of the essential proteins results in a more organized pattern of fluorescence typi-cal of DNA replication compartments.

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3. Reeves, R., Gorman, C., and Howard, B. (1985) Minichromosomes assembly ofnonintegrated plasmid DNA transfected into mammalian cells. Nucleic Acids Res.13, 3599–3605.

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7. Rupprecht, A. P. and Coleman, D. L. (1991) Transfection of adherent murine peri-toneal macrophages with a reporter gene using DEAE-dextran. J. Immunol. Meth-ods 144, 157–63.

8. Gauss, G. H. and Lieber, M. R. (1992) DEAE-dextran enhances electroporation ofmammalian cells. Nucleic Acids Res. 20, 6739–6740.

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10. Segura, I., Gonzalez, M. A., Serrano, A., Abad, J. L., Bernad, A., and Riese, H. H.(2001) High transfection efficiency of human umbilical vein endothelial cells usingan optimized calcium phosphate method. Anal. Biochem. 296, 143–147.

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13. Pari, G. S., Kacica, M. A., and Anders, D. G. (1993) Open reading frames UL44,IRS1/TRS1, and UL36-38 are required for transient complementation of human cy-tomegalovirus oriLyt-dependent DNA synthesis. J. Virol. 67, 2575–2582.

32 Pari and Xu